Modified Project Summary/Abstract Section CRISPR-Cas9 is revolutionizing the life sciences through its RNA-guided ability to target individual genes for disruption or for precise editing. In addition to accelerating basic research and biotechnology, Cas9 and its RNA guides also have the potential to transform the treatment of inherited diseases via genome editing-based cures, especially if delivery hurdles can be overcome and clinical safety can be proven. There are three subtypes of Cas9 systems (Types II-A, -B and -C), and all three have yielded Cas9 orthologs with demonstrated utility in mammalian genome editing. Our own work has established the Type II-C Cas9 from N. meningitidis (NmeCas9) as a compact, naturally hyperaccurate genome editing platform. Some of the safety concerns in Cas9?s clinical development arise from Cas9 activity that is excessive, prolonged, or present in unintended cell types. These concerns have been heightened by a shortage of effective tools that inactivate Cas9 proteins after completion of the intended editing, or that prevent Cas9 activity altogether at undesired times or in unintended tissues. We recently discovered natural Cas9 inhibitors [anti-CRISPR (Acr) proteins] that evolved as counter-measures against CRISPR immunity. We have identified and validated five distinct families of Acrs from bacterial species with Type II-C CRISPR-Cas systems, and established their efficacy as off-switches for NmeCas9 genome editing in human cells. Our proven strategies have also led us to additional Acr candidates that are likely inhibitors of distinct Type II-C Cas9 orthologs. Our discoveries therefore provide us with a powerful entre into addressing the limitations and safety concerns that arise from uncontrolled or otherwise unwanted Cas9 activity. In this proposal, we outline experiments that exploit our discovery of Cas9 inhibitors in three ways. In Aim 1, we will use Acr phylogenetic distributions as pointers towards orthologous, robust, compact Type II-C Cas9 genome-editing systems, each of which would be immediately amenable to off-switch control. Because distinct Cas9s often have novel targeting specificities, this work also promises to expand the genomic scope of Cas9 editing. In Aim 2, we will optimize Acr inhibitory potency, and test the hypothesis that Acrs can be used to increase the efficiency of precise editing via homology-dependent repair. Finally, in Aim 3, we will establish tissue-specific Acr control over genome editing in cultured mammalian cells and use it to develop a flexible platform for restricting Cas9 genome editing to a single desired cell type. Our strategy for enforcing tissue specificity of gene editing will be applicable even to cell types in which tissue-specific promoters are unavailable for driving Cas9 expression. The proposed research promises to yield enhanced genome-editing systems with broader targeting range, fewer safety risks and improved tissue specificity, which we will demonstrate pre-clinically in mammalian cells.